LOCUS       sequence-468436-        7666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent transfection marker encoding mCherry under PGK promoter 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7666)
  AUTHORS   Wolfsberg E, Paul J-P, Tycko J, Chen B, Bassik MC, Bintu L, Alizadeh
            AA, Gao XJ
  TITLE     Machine-Guided Dual-Objective Protein Engineering for Deimmunization
            and Therapeutic Functions
  JOURNAL   bioRxiv 2025.02.24.639918
REFERENCE   2  (bases 1 to 7666)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.02.24.639918"
COMMENT     Sequence Label: PGK-mCherry
FEATURES             Location/Qualifiers
     source          1..7666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1291..2313)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1291..2313)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    complement(2321..2368)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     2500..2519
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2691..2915
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(2977..3006)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(3022..3735)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYKS"
     primer_bind     complement(3303..3322)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3572..3590
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3742..4122)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MHPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYK
                     VLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLL
                     PGELAKHAVSEGTKAITKYTSAK"
     promoter        complement(4139..4638)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4187..4206)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4580..4603
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(4653..4686)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    4698..4905
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    5400..5521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5612..5636)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5646..5664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5667..5685
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(5687..5890)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5716..5736)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5891..6270
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6437..6456
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6531..6635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6636..6704,6705..7496)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6854..6873)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtagtacgtg ctattccttt gccctcggac gagtgctggg
     1321 gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct
     1381 tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
     1441 tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg
     1501 gtcaagacca atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg
     1561 cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa
     1621 gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc
     1681 tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
     1741 ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga
     1801 cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc
     1861 gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa
     1921 cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atggcctccg cgaccggctg
     1981 cagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
     2041 ggagatgcaa taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
     2101 gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca
     2161 gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc
     2221 ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt
     2281 ctcgacagac gtcgcggtga gttcaggctt tttggccaag gaagttccta tactttctag
     2341 agaataggaa cttcggaata ggaacttcta ggtacgtgaa ccatcaccct aatcaagttt
     2401 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2461 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2521 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2581 gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc
     2641 cgcctcagaa gccatagagc ccaccgcatc cccagaacat cgatagatct ccatagagcc
     2701 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     2761 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     2821 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     2881 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
     2941 tcaatgatga ttatgatgat ggtcgacggc gctattcaga tcctcttctg agatgagttt
     3001 ttgttcgggc cctcctcgag cttaagactt gtacagctcg tccatgccgc cggtggagtg
     3061 gcggccctcg gcgcgttcgt actgttccac gatggtgtag tcctcgttgt gggaggtgat
     3121 gtccaacttg atgttgacgt tgtaggcgcc gggcagctgc acgggcttct tggccttgta
     3181 ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc agcttcagcc tctgcttgat
     3241 ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct cccagcccat
     3301 ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt
     3361 gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca cgccgccgtc
     3421 ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct tcaagtagtc
     3481 gggaatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact gaggggacag
     3541 gatgtcccag gcgaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt
     3601 gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga
     3661 gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt tatcctcctc
     3721 gcccttgctc accatcctag gcttagcgct ggtgtacttg gtgatggcct tagtaccctc
     3781 ggacacggcg tgcttggcca actccccagg cagcagcagg cgcacggccg tctggatctc
     3841 cctggaggtg atggtcgagc gcttgttgta atgcgccagg cgggaagcct cacctgcgat
     3901 gcgctcgaaa atgtcgttca caaacgaatt catgatgccc atggccttgg acgaaatgcc
     3961 ggtgtcaggg tggacctgct tcagaacctt gtacacatag atggaatagc tctccttgcg
     4021 gctgcgcttg cgcttcttgc cgcctttctt ctgcgcctta gtcaccgcct tcttggagcc
     4081 ctttttcggg gcgggagcag acttcgctgg ctctgggtgc atggtggcga ctgcaggtcg
     4141 aaaggcccgg agatgaggaa gaggagaaca gcgcggcaga cgtgcgcttt tgaagcgtgc
     4201 agaatgccgg gcctccggag gaccttcggg cgcccgcccc gcccctgagc ccgcccctga
     4261 gcccgccccc ggacccaccc cttcccagcc tctgagccca gaaagcgaag gagcaaagct
     4321 gctattggcc gctgccccaa aggcctaccc gcttccattg ctcagcggtg ctgtccatct
     4381 gcacgagact aatgagacgt gctacttcca tttgtcacgt cctgcacgac gcgagctgcg
     4441 gggcgggggg gaacttcctg actaggggag gagtagaagg tggcgcgaag gggccaccaa
     4501 agaacggagc cggttggcgc ctaccggtgg atgtggaatg tgtgcgaggc cagaggccac
     4561 ttgtgtagcg ccaagtgccc agcggggctg ctaaagcgca tgctccagac tgccttggga
     4621 aaagcgcctc ccctacccgg tagagcggcc gcataacttc gtatagcata cattatacga
     4681 agttatgcta gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4741 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4801 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4861 cagcaagggg gaggattggg aagacaatag caggcatgct ggggaagata tagggacagc
     4921 ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga
     4981 gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc
     5041 ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc
     5101 tgctctttga gcctgcagac acctgggggg atacggggaa aaagccatac caatgggccc
     5161 taaaaaagga atccagtcaa ttccggggct aaacctggct gccactgttt ctttagggac
     5221 ttcgttcctg tgaggacagg aaaccctgca acctggttga ccagagcaag ggatataaga
     5281 acccagtttg ggaacactgc tgtttaaata ttaaacaggg atgtcttgtc ccccggaagg
     5341 gagaccagat gagcttctga tagactaatt gacatcattt gagtcaattc cagacatgat
     5401 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     5461 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     5521 taagcttaag tttaaacgct agagtccgga ggctggatcg gtcccggtgt cttctatgga
     5581 ggtcaaaaca gcgtggatgg cgtctccagg cgatctgacg gttcactaaa cgagctcgtc
     5641 gacgatctct atcactgata gggagatctc tatcactgat agggagagct ctgcttatat
     5701 agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac
     5761 attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt
     5821 ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa
     5881 accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt
     5941 cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa
     6001 tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta
     6061 aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg
     6121 tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg
     6181 taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt
     6241 actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt acgaagcagc
     6301 gcaaaacgcc taaccctaag cagattcttc atgcaattgt cggtcaagcc ttgccttgtt
     6361 gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga
     6421 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg
     6481 ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6541 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6601 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6661 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6721 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6781 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6841 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6901 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6961 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     7021 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     7081 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     7141 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     7201 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7261 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7321 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     7381 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7441 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7501 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7561 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7621 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
